Mutations E44D and V118I in the Reverse Transcriptase of HIV-1 Play Distinct Mechanistic Roles in Dual Resistance to AZT and 3TC

Retroviral resistance to AZT and 3TC has been associated with two different mechanisms. The M184V mutation in the reverse transcriptase (RT) of the human immunodeficiency virus, type 1 (HIV-1) diminishes the incorporation of 3TC-monophosphate (3TC-MP), whereas AZT resistance-conferring mutations were shown to facilitate the phosphorolytic excision of incorporated AZT-MP in the presence of ATP. Both mechanisms show a certain degree of incompatibility; however, previous clinical data revealed that mutations E44D and V118I, when present in a background of classical AZT mutations (M41L, D67N, L210W, and T215Y), confer dual resistance to AZT and 3TC. We have purified RT enzymes that contain E44D and V118I either alone or in a background of different combinations of AZT mutations to study the underlying biochemical mechanisms. We found that enzymes containing E44D in a background of these latter mutations increase the efficiency of excision of 3TC-MP. Unexpectedly, V118I-containing enzymes show dramatic reductions in rates of incorporation of AZT-MP and 3TC-MP. The V118I mutant is also associated with diminished rates of ATP-dependent primer unblocking. The additional presence of mutations M41L, D67N, L210W, and T215Y can partially neutralize this deficit, which helps to explain the concurrent presence of these changes in resistant isolates. These biochemical data make clear that mutations E44D and V118I play distinct mechanistic roles in dual resistance to AZT and 3TC. Our findings are consistent with an increasing number of clinical studies suggesting that the V118I cluster constitutes a novel pathway for HIV resistance to multiple nucleotide analogue RT inhibitors. Retroviral resistance to AZT and 3TC has been associated with two different mechanisms. The M184V mutation in the reverse transcriptase (RT) of the human immunodeficiency virus, type 1 (HIV-1) diminishes the incorporation of 3TC-monophosphate (3TC-MP), whereas AZT resistance-conferring mutations were shown to facilitate the phosphorolytic excision of incorporated AZT-MP in the presence of ATP. Both mechanisms show a certain degree of incompatibility; however, previous clinical data revealed that mutations E44D and V118I, when present in a background of classical AZT mutations (M41L, D67N, L210W, and T215Y), confer dual resistance to AZT and 3TC. We have purified RT enzymes that contain E44D and V118I either alone or in a background of different combinations of AZT mutations to study the underlying biochemical mechanisms. We found that enzymes containing E44D in a background of these latter mutations increase the efficiency of excision of 3TC-MP. Unexpectedly, V118I-containing enzymes show dramatic reductions in rates of incorporation of AZT-MP and 3TC-MP. The V118I mutant is also associated with diminished rates of ATP-dependent primer unblocking. The additional presence of mutations M41L, D67N, L210W, and T215Y can partially neutralize this deficit, which helps to explain the concurrent presence of these changes in resistant isolates. These biochemical data make clear that mutations E44D and V118I play distinct mechanistic roles in dual resistance to AZT and 3TC. Our findings are consistent with an increasing number of clinical studies suggesting that the V118I cluster constitutes a novel pathway for HIV resistance to multiple nucleotide analogue RT inhibitors. Nucleoside analogue reverse transcriptase inhibitors (NRTIs) 1The abbreviations used are: NRTI, nucleoside analogue reverse transcriptase inhibitor; RT, reverse transcriptase; HIV-1, human immunodeficiency virus type 1; AZT, 3′-azido-3′-deoxythymidine; D4T, 2′,3′-didehydro-2′,3′-dideoxythymidine; ddI, 2′,3′-dideoxyinosine; 3TC, (-)-β-l-2′,3′-dideoxy-3′-thyacytidine; ddC, 2′,3′-dideoxycytidine; TP, triphosphate; MP, monophosphate; WT, wild-type; NAM, nucleoside-associated mutation.1The abbreviations used are: NRTI, nucleoside analogue reverse transcriptase inhibitor; RT, reverse transcriptase; HIV-1, human immunodeficiency virus type 1; AZT, 3′-azido-3′-deoxythymidine; D4T, 2′,3′-didehydro-2′,3′-dideoxythymidine; ddI, 2′,3′-dideoxyinosine; 3TC, (-)-β-l-2′,3′-dideoxy-3′-thyacytidine; ddC, 2′,3′-dideoxycytidine; TP, triphosphate; MP, monophosphate; WT, wild-type; NAM, nucleoside-associated mutation. represent an important class of compounds to treat infection with the human immunodeficiency virus, type 1 (HIV-1). Seven different NRTIs are in clinical use: 3′-azido-3′-deoxythymidine (zidovudine or AZT), 2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine or D4T), 2′,3′-dideoxyinosine (didanosine or ddI), (-)-β-l-2′,3′-dideoxy-3′-thyacytidine (lamivudine or 3TC), 2′,3′-dideoxycytidine (zalcitabine or ddC), and, more recently, the carboxylic nucleoside abacavir, and the acyclic inhibitor tenofovir. These compounds are intracellularly phosphorylated and compete with natural dNTP pools for incorporation into the growing DNA chain. The relevant metabolites of ddI and abacavir are ddATP and carbovir triphosphate, respectively. Although the clinical use of these chain terminators in combination with non-nucleoside analogue RT inhibitors (non-NRTIs), viral protease inhibitors (PIs), can sometimes reduce the viral burden to levels below the limit of detection, the development of drug resistance cannot be prevented. The emergence of resistance-conferring mutations is a major cause for viral rebound and treatment failure, and certain mutational patterns are associated with decreased susceptibility to multiple NRTIs, which can dramatically reduce remaining options for the management of the infection. Here we studied reaction pathways involved in dual resistance to AZT and 3TC. Both drugs are frequently administered together in combination with a non-NRTI or a protease inhibitor. Recent studies have shown that resistance to the individual drugs involves different biochemical mechanisms that show a certain degree of incompatibility (1Larder B.A. Kemp S.D. Harrigan P.R. Science. 1995; 269: 696-699Crossref PubMed Scopus (622) Google Scholar, 2Gotte M. Wainberg M.A. Drug Resist Updat. 2000; 3: 30-38Crossref PubMed Scopus (30) Google Scholar). The M184V mutation confers high level (>100-fold) resistance to 3TC (3Schinazi R.F. Lloyd Jr., R.M. Nguyen M.H. Cannon D.L. McMillan A. Ilksoy N. Chu C.K. Liotta D.C. Bazmi H.Z. Mellors J.W. Antimicrob. Agents Chemother. 1993; 37: 875-881Crossref PubMed Scopus (344) Google Scholar, 4Boucher C.A. Cammack N. Schipper P. Schuurman R. Rouse P. Wainberg M.A. Cameron J.M. Antimicrob. Agents Chemother. 1993; 37: 2231-2234Crossref PubMed Scopus (295) Google Scholar) (5Gao Q. Gu Z. Parniak M.A. Cameron J. Cammack N. Boucher C. Wainberg M.A. Antimicrob. Agents Chemother. 1993; 37: 1390-1392Crossref PubMed Scopus (214) Google Scholar). This mutation caused reductions in rates of incorporation of 3TC-MP, whereas rates of incorporation of dCTP remained largely unaffected (6Krebs R. Immendorfer U. Thrall S.H. Wohrl B.M. Goody R.S. Biochemistry. 1997; 36: 10292-10300Crossref PubMed Scopus (135) Google Scholar). In contrast, mutant enzymes containing classical AZT-associated amino acid substitutions, i.e. M41L, D67N, K70R, L210W, T215(F/Y), and K219Q, were shown to incorporate AZT-MP with similar efficiency as seen with wild-type RT (6Krebs R. Immendorfer U. Thrall S.H. Wohrl B.M. Goody R.S. Biochemistry. 1997; 36: 10292-10300Crossref PubMed Scopus (135) Google Scholar, 7Lacey S.F. Reardon J.E. Furfine E.S. Kunkel T.A. Bebenek K. Eckert K.A. Kemp S.D. Larder B.A. J. Biol. Chem. 1992; 267: 15789-15794Abstract Full Text PDF PubMed Google Scholar). However, both wild-type HIV-1 RT and AZT-resistant mutant enzymes are capable of removing the incorporated nucleotide analogue in the presence of physiologically relevant concentrations of pyrophosphate or ATP (8Arion D. Kaushik N. McCormick S. Borkow G. Parniak M.A. Biochemistry. 1998; 37: 15908-15917Crossref PubMed Scopus (313) Google Scholar, 9Meyer P.R. Matsuura S.E. So A.G. Scott W.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13471-13476Crossref PubMed Scopus (300) Google Scholar). The latter was shown to act as a pyrophosphate donor. Rates of ATP-dependent rescue of chain-terminated DNA synthesis are significantly increased with mutant enzymes containing AZT resistanceconferring mutations, which provides an important mechanism for resistance to the drug (10Meyer P.R. Matsuura S.E. Mian A.M. So A.G. Scott W.A. Mol. Cell. 1999; 4: 35-43Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 11Meyer P.R. Matsuura S.E. Tolun A.A. Pfeifer I. So A.G. Mellors J.W. Scott W.A. Antimicrob. Agents Chemother. 2002; 46: 1540-1545Crossref PubMed Scopus (67) Google Scholar). Combinations of at least two to three of the classical AZT mutations can also reduce susceptibility to other NRTIs. Accordingly, these amino acid substitutions have been named nucleoside-associated mutations (NAMs). Biochemical studies have shown that corresponding mutant enzymes can indeed increase rates of excision of AZT-MP, D4T-MP, ddAMP, and carbovir monophosphate (12Naeger L.K. Margot N.A. Miller M.D. Antimicrob. Agents Chemother. 2002; 46: 2179-2184Crossref PubMed Scopus (88) Google Scholar). The rate of excision depends on both the chemical nature of the chain terminator and the specific mutational pattern. Unblocking of AZT-terminated primers is relatively efficient, whereas the removal of 3TC-MP is difficult to detect. We have demonstrated previously (13Gotte M. Arion D. Parniak M.A. Wainberg M.A. J. Virol. 2000; 74: 3579-3585Crossref PubMed Scopus (161) Google Scholar) that M184V-containing mutant enzymes are literally unable to remove 3TC-MP, and the rescue of AZT-terminated DNA synthesis was also compromised in this context. These results help to explain previous tissue culture and clinical data showing that the M184V mutation can increase susceptibility to AZT, which can sometimes lead to resensitization of formerly AZT-resistant viruses (1Larder B.A. Kemp S.D. Harrigan P.R. Science. 1995; 269: 696-699Crossref PubMed Scopus (622) Google Scholar). Such phenotypes are of potential clinical benefit; however, drug hypersusceptibility and resensitization effects may occur only transiently, as shown for AZT/3TC combination therapy (14Miller V. Phillips A. Rottmann C. Staszewski S. Pauwels R. Hertogs K. de Bethune M.P. Kemp S.D. S. Harrigan P.R. Larder B.A. J. 1998; PubMed Scopus Google Scholar). The resistant is seen in the of clinical resistance is found in the of The which is with changes at and confers resistance to multiple NRTIs AZT and 3TC. of i.e. or and in the RT is also associated with decreased susceptibility to a of NRTIs B.A. S. Kemp S.D. Hertogs K. Miller V. M. Staszewski S. J. Pauwels R. Antimicrob. Agents Chemother. 1999; PubMed Google Scholar). However, the of of these two patterns is relatively clinical with or high level resistance to 3TC K. K. J. Boucher C.A. M. J. C. C. A. Schuurman R. J. S. M. S. J. A.M. Antimicrob. Agents Chemother. 2000; PubMed Scopus Google Scholar). the associated with susceptibility to both AZT and 3TC are mutations E44D or V118I in with two or more C. M. A. A. J.M. C. V. Antimicrob. Agents Chemother. PubMed Scopus Google Scholar, C. A. V. J. 2000; PubMed Scopus Google Scholar, K. S. V. C. P. A. M. S. M. Miller V. Larder B.A. Antimicrob. Agents Chemother. 2000; PubMed Scopus Google Scholar, J. M.P. N. J. D. A. N. R. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, M. J. Virol. 2002; PubMed Scopus Google Scholar, G. S. Harrigan R. Larder B.A. M. J. 2002; PubMed Scopus Google Scholar, S. J. C. J. D. Antimicrob. Agents Chemother. 2002; 46: PubMed Scopus Google Scholar). These mutations were found in showing levels of resistance to 3TC in the of M184V K. S. V. C. P. A. M. S. M. Miller V. Larder B.A. Antimicrob. Agents Chemother. 2000; PubMed Scopus Google Scholar). Recent of HIV treatment and resistance a of and G. S. Harrigan R. Larder B.A. M. J. 2002; PubMed Scopus Google Scholar). The of these mutations is significantly increased in as with treatment of The V118I mutation has also been in the of drug combinations containing AZT G. Arion D. Wainberg M.A. Parniak M.A. Antimicrob. Agents Chemother. 1999; PubMed Google Scholar). The previous showing that dual resistance to AZT and 3TC the additional presence of at least two classical that E44D or and V118I effects a mechanism that may ATP-dependent excision of both 3TC-MP and this we mutant enzymes that the two mutations alone or together in a background of combinations of and studied these mutant enzymes in to to incorporate and to AZT-MP and 3TC-MP, respectively. Unexpectedly, we found that and play distinct roles in HIV resistance to nucleotide The rates of excision of 3TC-MP, whereas the latter diminishes the excision of chain terminators and decreased rates of incorporation of multiple nucleotide AZT-MP and 3TC-MP. and HIV-1 RT and mutant enzymes were and purified as previously S.F. J. PubMed Scopus Google Scholar). were in previous studies (13Gotte M. Arion D. Parniak M.A. Wainberg M.A. J. Virol. 2000; 74: 3579-3585Crossref PubMed Scopus (161) Google Scholar, M. M. N. Wainberg M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we the as as a DNA and and were used as primer of the primer and were M. M. N. Wainberg M.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The i.e. are and respectively. was R. C. was and were and and are and respectively. of DNA with effects of were with wild-type HIV-1 RT and mutant enzymes use of of RT or of of and and concentrations of and in and DNA synthesis was the of were to for at and with of were and the remaining was were at concentrations of and concentrations of The data were and the The of the was J. K. P. S.H. Biochemistry. PubMed Scopus Google Scholar). were in either the or presence of ATP to the of changes that both the incorporation and the excision of the inhibitor. of ATP were with of in a containing 1 to the of was as previously (13Gotte M. Arion D. Parniak M.A. Wainberg M.A. J. Virol. 2000; 74: 3579-3585Crossref PubMed Scopus (161) Google Scholar). of the was with of HIV-1 RT in a containing the of of the and concentrations of were with and to for at were with and on were or use of a were as previously R.F. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Unblocking and of DNA and the rescue of chain-terminated DNA synthesis was in the of ATP and the dNTP as in the were at and at different of reaction to of were for at and on distinct AZT resistance pathways have been in J. 2000; PubMed Scopus Google Scholar). T215Y is found in a background of and L210W, and is found in background of and clinical studies have shown that mutations E44D or and occur in a background of mutations that to the pathway (M41L, D67N, L210W, and G. S. Harrigan R. Larder B.A. M. J. 2002; PubMed Scopus Google Scholar, J. 2000; PubMed Scopus Google Scholar). We biochemical study on the of the two mutations, E44D and on of DNA with study the of these mutations on biochemical mechanisms involved in dual resistance to AZT and 3TC, we RT enzymes that mutations E44D or V118I either alone or together with different combinations of amino acid substitutions at and These enzymes were for to the effects of The were in the and in the presence of ATP to the of changes in to both incorporation and excision of the mutant enzymes are as as with wild-type HIV-1 RT, on the of both and In with previous we found that enzymes with classical (M41L, D67N, L210W, and show to in when were in the presence of ATP P.R. Matsuura S.E. Tolun A.A. Pfeifer I. So A.G. Mellors J.W. Scott W.A. Antimicrob. Agents Chemother. 2002; 46: 1540-1545Crossref PubMed Scopus (67) Google Scholar). effects are seen with an containing mutations and T215Y together with the J. Hertogs K. Larder B.A. J. Virol. PubMed Scopus Google Scholar, A. M. C. V. M.A. J. 2000; PubMed Scopus Google Scholar). either alone or in a background of classical to levels of ATP-dependent primer unblocking. of these enzymes show effects in the of of DNA synthesis in the presence of wild-type HIV-1 RT resistant mutant are of three to are a of resistance is in are of three to are a of resistance is in in a The level of of DNA synthesis is dramatically decreased with in the of ATP to Both and similar The data that V118I, classical resistance-conferring effects and natural mutant enzymes containing both V118I and the classical 3TC resistance-conferring mutation M184V similar that the of M184V on rates of excision of AZT-MP may be the diminished rates of incorporation of the chain We in associated with mutant however, with to wild-type RT are These results that the are significantly this mutation. in the E44D V118I M184V-containing in to the associated with M184V-containing enzymes have been (6Krebs R. Immendorfer U. Thrall S.H. Wohrl B.M. Goody R.S. Biochemistry. 1997; 36: 10292-10300Crossref PubMed Scopus (135) Google Scholar). However, the data also In to tissue culture and clinical results show that V118I in the of can the effects of the of E44D is these and E44D and susceptibility to 3TC. of E44D and V118I on the of of these we and the the use of DNA synthesis was in the presence of increasing concentrations of to the of the mutant enzymes to the nucleotide analogue and natural a RT and the V118I mutant as an The results are in with the data shown in I. We found that the mutant diminishes the efficiency of chain whereas the mutant is the wild-type in this revealed to in to the efficiency of incorporation of AZT-MP associated with V118I The is when an that V118I in a background of which is also the results with the of chain of different are of two to three are a of are of two to three are a of in a on the of of studied the of the mutant enzymes to the incorporated AZT-MP the primer The different enzymes were with a that an AZT-terminated primer and a of ATP and to primer and to the of three more The rescue of chain-terminated DNA synthesis was in We found that the ATP-dependent of the AZT-terminated primer and the of DNA synthesis is compromised with the whereas the mutant show when with the RT and the V118I mutation the incorporation of AZT-MP, as as This provides a for clinical and culture studies that show in susceptibility to AZT in a of viruses that contain the V118I that the diminished efficiency of primer may neutralize the effects of the diminished rates of incorporation of with data we show that containing mutant can increase levels of rescue of DNA The additional presence of to increase or this and However, the presence of can the of enzymes to rescue AZT-terminated DNA the efficiency of the reaction is diminished as with wild-type RT and that resistance-conferring effects at the level of and the background of is to for in to the ATP-dependent on Both and of efficiency of chain with was studied the as in the and of for AZT-MP, we found that V118I decreased the efficiency of incorporation of 3TC-MP whereas the E44D mutant RT of the data revealed in with the mutant and an that in a background of together with E44D We the M184V mutant in this and a increase in the and These data are in with viral phenotypes that show high level resistance to 3TC associated with M184V and levels of 3TC resistance associated with However, at the is important to that the diminished viral susceptibility to 3TC is associated with the additional presence of at least two or three as for AZT, is that reductions in rates of excision may neutralize the diminished rates of incorporation of The of the efficiency of these however, the that the removal of 3TC-MP is compromised with study rescue of DNA we used an as in the ATP-dependent rescue of DNA synthesis is with the wild-type the V118I and the mutant and Rates of rescue are increased in the presence of the and a mutant containing in a background of caused a increase in the efficiency of removal of 3TC-MP and the rescue of DNA synthesis and Such an increase is the clear that can be to the E44D mutation. Although the efficiency of the reaction is as as seen with AZT-terminated primers the increase in rates of excision may be to levels of resistance to the drug as seen in clinical This is the that the efficiency of incorporation of 3TC-MP is relatively in that DNA synthesis may to be frequently The additional presence of V118I can reverse this which the of this mutation on the excision reaction and on the and of we also in this We found that the efficiency of chain with is also diminished with mutant We a to increase in as with wild-type HIV-1 RT levels of decreased rates of incorporation have also been with the other We the efficiency of excision of with mutant enzymes containing together with and together with and The results are consistent with seen with primer The caused an increase in rescue of DNA and the V118I in the mutational background can partially reverse this HIV resistance in the presence of E44D and V118I may be to AZT and 3TC. The data make clear that both mutations play distinct mechanistic the can increase rates of excision in the presence of the latter the nucleotide analogue inhibitors and natural dNTP use of drugs can in mutational patterns and viral phenotypes that resistance to multiple and, on the other of the drug hypersusceptibility and resensitization The study of biochemical mechanisms associated with changes in drug susceptibility an important in to resistance-conferring Here we studied biochemical mechanisms associated with dual resistance to AZT and 3TC mutations E44D and We mutant enzymes in to to incorporate and to AZT-MP and 3TC-MP. changes in both of as with the wild-type can susceptibility to a However, the and viral phenotypes with an increasing number of mutations that may effects on the of HIV-1 facilitate a with viral we biochemical data in The results with to the efficiency of both incorporation and excision of chain terminators are in three and In with previous studies (10Meyer P.R. Matsuura S.E. Mian A.M. So A.G. Scott W.A. Mol. Cell. 1999; 4: 35-43Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 11Meyer P.R. Matsuura S.E. Tolun A.A. Pfeifer I. So A.G. Mellors J.W. Scott W.A. Antimicrob. Agents Chemother. 2002; 46: 1540-1545Crossref PubMed Scopus (67) Google we found that different combinations of M41L, D67N, L210W, and T215Y facilitate the ATP-dependent of AZT-terminated These results that the excision of incorporated AZT-MP is a major in viral resistance to AZT, whereas to be in this Here we show that resistance to AZT can both reaction and the presence or of a amino acid i.e. V118I, can or increased rates of excision of the chain terminator is in this The V118I mutation alone the efficiency of both incorporation and excision of The to natural dNTP pools and the nucleotide analogue at the level of or incorporation susceptibility to however, a in efficiency of excision is to this that the effects of reductions of both incorporation and excision of AZT-MP in this which helps to explain mutant viruses that contain alone are associated with changes in drug The viral on the additional presence of that levels of ATP-dependent primer unblocking. However, data that is the mechanism associated with V118I, the efficiency of excision increased the levels seen with The may also help to explain viruses containing in a background of both and have been associated with AZT resensitization effects K. S. V. C. P. A. M. S. M. Miller V. Larder B.A. Antimicrob. Agents Chemother. 2000; PubMed Scopus Google Scholar, G. S. Harrigan R. Larder B.A. M. J. 2002; PubMed Scopus Google Scholar, M. K. Antimicrob. Agents Chemother. 2002; 46: PubMed Scopus Google of the latter two mutations can reduce the efficiency of rescue of AZT-terminated DNA that the reductions with to the incorporation of AZT-MP are to for these Although the biochemical data are consistent with drug susceptibility in this may be difficult to the of both on the in other the rates of nucleotide incorporation are significantly as with rates of is that viruses containing V118I in the of may also be associated with hypersusceptibility to other that the diminished rates of excision are diminished rates of incorporation of the inhibitor. V118I-containing enzymes are also associated with diminished rates of incorporation of 3TC-MP, which provides a mechanism for dual resistance to AZT and 3TC. In contrast, enzymes are literally RT in this however, this mutation caused in rates of excision of incorporated 3TC-MP, when present in a background of dual resistance to AZT and 3TC is in this latter on ATP-dependent unblocking. for AZT, the additional presence of diminishes the excision and the in which is also consistent with culture data showing that E44D and V118I to or effects G. S. Harrigan R. Larder B.A. M. J. 2002; PubMed Scopus Google Scholar). these data make clear that mutations E44D and V118I are and play distinct roles in dual resistance to AZT and 3TC. These findings are in with the different of and in the of HIV-1 RT R. Science. 1998; PubMed Scopus Google Scholar). V118I in to and which with the at these were shown to have effects on the of RT to and as as and pools A.M. J. PubMed Scopus Google Scholar, S.H. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). mutation to the cluster that is associated with resistance to multiple NRTIs. J. J. M.P. C. S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) have shown that this mutational effects at the level of nucleotide The that the cluster the and the may a similar in the of The chain may the of to the dNTP In contrast, is the dNTP however, the classical AZT-associated mutation M41L, is in to the of ATP S.H. J. Virol. PubMed Scopus Google Scholar, J. J. Larder B.A. J. Virol. 2002; PubMed Scopus Google Scholar). is that E44D can the the pyrophosphate and the studies that the development of the two different mechanisms associated with resistance to NRTIs may be the different chemical nature of the nucleotide and with RT C. C. P. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The excision mechanisms in the of that are the wild-type and in the of that are and M184V and 3TC. This may for treatment that a however, the development of resistance to multiple drugs to The of E44D that the of additional mutations can the excision mechanism and this pathway for an increasing number of nucleotide that are 3TC-MP and the results with enzymes show that an can play an important at the The biochemical data in this study are consistent with an increasing number of clinical studies suggesting that E44D and V118I, when present in a background of are associated with resistance to multiple NRTIs M. J. Virol. 2002; PubMed Scopus Google Scholar, G. S. Harrigan R. Larder B.A. M. J. 2002; PubMed Scopus Google Scholar, M. K. Antimicrob. Agents Chemother. 2002; 46: PubMed Scopus Google Scholar). We A. Wainberg for

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